Capturing cold-stress-related sequence diversity from a wild relative of common bean (Phaseolus angustissimus)

One restriction to the cultivation of common bean, Phaseolus vulgaris L., is its limited tolerance to low temperatures. In the present study, subtraction suppression hybridization was employed to enrich for stress responsive genes in both a chilling-susceptible common bean and a relatively more chilling-tolerant wild bean species, Phaseolus angustissimus. For each species, approximately 11 000 expressed sequence tags were generated. Comparative sequence analysis of the EST collection with the available annotated genome sequences of the model Fabaceae species Medicago truncatula and Glycine max identified protein homologues for approximately 65% and 80% of the Phaseolus sequences, respectively. This difference reflects the closer phylogenetic relationship between the genera Phaseolus and Glycine compared with Medicago. Annotation of the Phaseolus sequences was facilitated through this comparative analysis and indicated that several heat shock proteins, cytochrome P450s, and DNA binding factors were uniquely found among the sequences from the wild species P. angustissimus. The Phaseolus sequences have been made available on a GBrowse implementation using M. truncatula as the reference genome, providing rapid access to the sequence data and associated comparative genome data.     

Phylogenetic study on wild allies of Lima bean,Phaseolus lunatus (Fabaceae), and implications on its origin

An investigation was made of the phylogenetic relationships among wild accessions of Lima bean (Phaseolus lunatus) and wild allies of Mesoamerican and Andean origins, using electrophoresis of seed storage proteins and isozymes. Mesoamerican wild species are phylogenetically more distant fromP. lunatus than Andean species, and apparently belong to the tertiary gene pool of Lima bean. The Andean wild species, which are investigated for the first time, reveal a high similarity to the Lima bean, and particularly with its Mesoamerican gene pool. These Andean species probably constitute a secondary gene pool of Lima bean, and are thus of considerable interest in the context of genetic improvement of the crop. Based on these observations, an Andean origin is suggested for the Andean wild species and forP. lunatus. These results point out the importance of collecting and conserving AndeanPhaseolus germplasm.     

Control of Paraheliotropism in Two Phaseolus Species

Paraheliotropic (light-avoiding) leaf movements have been associated with high light intensity, high temperature, and drought. We investigated leaf elevation for intact plants, pulvinus bending for excised motor organs, and size change for protoplasts from motor tissue for two Phaseolus species: Phaseolus acutifolius A. Gray, native to hot, arid regions, and Phaseolus vulgaris L., the common bean. Leaf angles above horizontal were measured for central trifoliolate leaflets of intact plants at 24, 27, and 30[deg]C at 500 and 750 [mu]mol photons (400-700 nm) m-2 s-1 over a range of water potentials; equivalent angles were determined for excised motor organs under similar conditions. Diameters were measured for protoplasts from abaxial and adaxial motor tissue over a range of photon flux density values, temperatures, and water potentials. In general, higher photon flux density and temperature resulted in elevation of leaves, bending of excised pulvini, and equivalent changes in protoplast volume (swelling of abaxial protoplasts and shrinking of adaxial protoplasts). In intact plants, lower water potentials yielded greater paraheliotropism; abaxial protoplasts increased in size, whereas adaxial ones did not change. P. acutifolius typically exhibited greater paraheliotropism than did P. vulgaris under the same conditions, a set of physiological responses likely to be highly adaptive in its native arid habitat.     

First use of microsatellite markers in a large collection of cultivated and wild accessions of tepary bean (Phaseolus acutifolius A. Gray)

Tepary bean (Phaseolus acutifolius A. Gray) is a dry-land crop species that originated in the deserts of Mexico and the south-western United States and therefore is proposed as a source of drought and salt tolerance for related species and for production in marginal rainfall areas. Few genetic tools have been developed or tested for tepary bean but microsatellites from common bean are an obvious choice for diversity analysis in the crop. The first goal of this study was to validate a set of gene-derived and non-gene simple sequence repeat or microsatellite markers from common bean in tepary bean cultivars and wild relative accessions. The second and more extensive objective of this study was to evaluate the genetic diversity and population structure of the tepary bean accessions to determine if leaf-morphology variants are valid as separate sub-groups of wild tepary beans; if P. parvifolius exist as a separate variants or species; and if cultivated tepary beans originated from one domestication event or several events. Our analysis of 140 tepary bean genotypes showed that a single domestication was likely as the cultivars were most closely related to accessions from Sinaloa and northern Mexico and that diversity was much higher in the wild genotypes compared to the cultivated ones. Other results were that P. parvifolius was classified as a separate species by population structure analysis while the variants P. acutifolius var. acutifolius and var. tenuifolius were admixed and inter-crossed. P. latifolius is not a valid species or variant of P. acutifolius but represents a group of cultivars within tepary bean. This is the first analysis of microsatellite diversity in tepary beans and has implications for breeding and conservation of this crop and its wild relatives.     

Frost resistance of winter rape leaves as related to the changes in water potential and growth capability

Differential thermal analysis indicated that the frost resistance of winter rape leaves ( Brassica napus L. var. oleifera L. cv. Gòrczanski), collected from plants grown in the cold (5/2°C), relies mainly on their ability to supercool to −9 to −11°C, i.e. consists in freezing avoidance. Initiation of ice formation in the cold-acclimated leaves resulted in the death of more than 50% of the cells as determined with a conductivity method. The development of freezing tolerance appeared to be an attribute of the second stage of plant hardening and was induced by the exposure of plants to a slightly subzero temperature (−5°C) for 18 h. Such a treatment brought about a sudden and persistent water potential decrease in the leaves, despite the fact that they had reabsorbed water from the medium prior to water potential measurements. Water potential changes were associated with a higher growth capability of the leaves as checked by determinations of disk area increments. It is suggested that the increased frost tolerance of the cold-grown winter rape leaves, subjected to subfreezing temperature, is related to the decreased water potential of the tissue caused by changes in turgor and/or in osmotic pressures of the cells.     

Effects of water stress on the water relations of Phaseolus vulgaris and the drought resistant Phaseolus acutifolius

The tepary bean ( Phaseolus acutifolius Gray var. latifolius ), a drought resistant species, was compared under water stress conditions with the more drought susceptible P. vulgaris L. cvs Pinto and White Half Runner (WHR). In order to better understand the basis for the superior drought resistance of tepary, this study was designed to determine the relationships among leaf water potential, osmotic potential, turgor potential, and relative water content (RWC).
Plants were prestressed by withholding irrigation water. These stress pretreatments changed the relation between leaf water potential and relative water content of both species so that prestressed plants had lower water potentials than controls at the same leaf RWC. Tepary had lower water potentials at given RWC levels than Pinto or WHR; this can account for part of the superior resistance of tepary. In all genotypes, prestressed plants maintained osmotic potentials approximately 0.2 MPa lower than controls. Tepary reached osmotic potentials that were significantly lower (0.15 to 0.25 MPa) than Pinto or WHR. Both control and prestressed tepary plants had 0.05 to 0.25 MPa more turgor than Pinto or WHR at RWC values between 65 and 80%. Both prestressed and control tepary plants had greater elasticity (a lower elastic modulus) than Pinto or WHR. This greater turgor of tepary at low RWC values could be caused by several factors including greater tissue elasticity, active accumulation of solutes, or greater solute concentration.
Tepary had significantly lower osmotic potentials than the P. vulgaris cultivars, but there was little difference in osmotic potential between Pinto and WHR. Knowledge of differences in osmotic and turgor potentials among and within species could be useful in breeding for drought resistance in Phaseolus.     

Cells with Crystals of Calcium Oxalate in the Leaves of Phaseolus vulgaris — a Comparison with those in Canavalia ensiformis

Leaflets of Phaseolus vulgaris contain crystals of calcium oxalate in the adaxial bundle sheath extensions. Most of the crystals accompany the lateral veins of third order. The average oxalate content of the leaves is 0.8% of dry weight. Some features of leaflet anatomy of Phaseolus and Canavalia are shown and the possible relation of anatomy to localization and development of crystals in each of the species is discussed. The majority of crystals in Phaseolus originate with the young leaflets newly unfolded. Calcium deficiency reduces number and size of crystals.     

Abscission as a mobilization phenomenon

The possibility that leaf abscission might involve a mobilization phenomenon leading to a localized cellular senescence was examined by following dry weight changes, chlorophyll content, and protein levels in proximal and distal tissue of excised pieces of bean petioles from Phaseolus vulgaris L. var. Red Kidney. Nucleic acid levels were determined as were phosphate pool sizes in conjunction with ³²P labeling patterns. Each of these parameters indicated a mobilization into the tissues proximal to the zone of cellular separation at the expense of the distal tissues, suggesting that mobilization and the consequent development of cellular senescence provide a component in the process of bean leaf abscission.     

Diversity in the rhizobia associated with Phaseolus vulgaris L. in Ecuador, and comparisons with Mexican bean rhizobia

Common beans (Phaseolus vulgaris L.) have centers of origin in both Mesoamerica and Andean South America, and have been domesticated in each region for perhaps 5000 years. A third major gene pool may exist in Ecuador and Northern Peru. The diversity of the rhizobia associated with beans has also been studied, but to date with an emphasis on the Mesoamerican center of origin. In this study we compared bean rhizobia from Mexico and Andean South America using both phenotypic and phylogenetic approaches. When differences between the rhizobia of these two regions were shown, we then examined the influence of bean cultivar on the most probable number (MPN) count and biodiversity of rhizobia recovered from different soils. Three clusters of bean rhizobia were distinguished using phenotypic analysis and principal-component analysis of Box AIR-PCR banding patterns. They corresponded principally to isolates from Mexico, and the northern and southern Andean regions, with isolates from southern Ecuador exhibiting significant genetic diversity. Rhizobia from Dalea spp., which are infective and effective on beans, may have contributed to the apparent diversity of rhizobia recovered from the Mesoamerican region, while the rhizobia of wild Phaseolus aborigineus from Argentina showed only limited similarity to the other bean rhizobia tested. Use of P. vulgaris cultivars from the Mesoamerican and Andean Phaseolus gene pools as trap hosts did not significantly affect MPN counts of bean rhizobia from the soils of each region, but did influence the diversity of the rhizobia recovered. Such differences in compatibility of host and Rhizobium could be a factor in the poor reputation for nodulation and N2 fixation in this crop.     

Regulation of alternative oxidase activity during phosphate deficiency in bean roots (Phaseolus vulgaris)

Cyanide-resistant respiration was studied in mitochondria isolated from the roots of bean plants ( Phaseolus vulgaris L. cv. Złota Saxa) grown hydroponically up to 16 days on a phosphate-sufficient (+P, control) or phosphate-deficient (−P) medium. Western blotting indicated that the alternative oxidase (AOX) was present only in its reduced (active) form, both in phosphate-sufficient and phosphate-deficient roots, but in the latter, the amount of AOX protein was greater. Addition of pyruvate to the isolation, washing and reaction media made mitochondria from +P roots cyanide-insensitive, similar to mitochondria from −P roots. The doubled activity of NAD-malic enzyme (NAD-ME) in −P compared with +P root mitochondria may suggest increased pyruvate production in −P mitochondria. Lower cytochrome c oxidase (COX) activity and no uncoupler effect on respiration indicated limited cytochrome chain activity in −P mitochondria. In −P mitochondria, the oxygen uptake decreased and the level of Q reduction increased from 60 to 80%. With no pyruvate present (AOX not fully activated), inhibition of the cytochrome pathway resulted in an increased level of the ratio of reduced ubiquinone (Qr) to total ubiquinone (Qt) (Qr/Qt) in +P mitochondria, but did not change Qr/Qt in −P mitochondria. When pyruvate was present, the kinetics for AOX were similar in mitochondria from −P and +P roots. It is suggested that AOX participation in −P respiration may provide an acclimation to phosphate deficiency. Stabilization of the ubiquinone reduction level by AOX might prevent the harmful effect of an increased formation of reactive oxygen species.     

Experimental studies on the drought, waterlogging, and frost tolerance of

Ascarina lucida Hook.f. (Chloranthaceae) is a small tree species endemic to New Zealand. The distribution of A. lucida suggests an inability to survive severe frosts or droughts. Therefore, peaks in the abundance of A. lucida in pollen records have usually been interpreted as indicating periods of mild, moist climates. The environmental tolerance of A. lucida seedlings to climatic extremes was experimentally tested by exposing seedlings to frost, drought, and waterlogged soil conditions. This research confirms the sensitivity of A. lucida to climatic extremes. Ascarina lucida has a similar drought tolerance to Coprosma grandifolia, a species known to be drought intolerant; seedlings had considerable tolerance of waterlogged soils, but exhibited reduced root weights when severely waterlogged; and a frost of -2°C resulted in complete mortality for seedlings sourced from lowland and submontane populations. Peaks in the abundance of A. lucida can be attributed, at least in part, to periods of warm, wet climate. However, the early successional nature of this species also suggests that disturbance regime plays an important role in regulating its distribution and abundance.     

RFLP diversity of common bean (Phaseolus vulgaris) in its centres of origin.

Eighty-five wild and cultivated accessions of common bean (Phaseolus vulgaris L.), representing a wide geographic area in the centres of domestication were tested for restriction fragment length polymorphisms (RFLPs). Genomic DNA was digested with one of three restriction enzymes (EcoRI, EcoRV, and HindIII) and hybridized to 12 probes distributed throughout the common bean genome. Accessions could be classified into two major groups with a distinct geographical distribution in Middle America and the Andes. Within each gene pool, cultivated accessions clustered together with wild forms from the same geographical area supporting the multiple domestications hypothesis for this crop. Estimates of Nei's genetic distances among the cultivated races from the two different gene pools varied from 0.12 to 0.56 and among races from the same gene pool from 0.04 to 0.12, suggesting that the divergence in Phaseolus vulgaris has reached the subspecies level. The level of genetic diversity (Ht = 0.38) was twice the value obtained with isozyme analysis. Genetic diversity within races (Hs = 0.27) was four to five times higher compared with isozymes, but genetic diversity between races (Dst = 0.11) was similar for both categories of markers. These results corroborate previous studies on the characterization of genetic diversity in common bean that clearly showed two distinct gene pools, Middle American and Andean. Moreover, RFLP markers are superior to isozymes because they provide better coverage of the genome and reveal higher level of polymorphisms.     

Nitrate reductase activity, distribution, and response to nitrate in two contrasting Phaseolus species inoculated with Rhizobium spp

The nitrate reductase activity distribution and response of two nodulated species of Phaseolus (Phaseolus vulgaris-common bean, and Phaseolus lunatus-lima bean) to different exogenous nitrate levels were studied during the vegetative period. These Phaseolus species showed to be very contrasting in respect to the pattern of nitrate reductase (NR) activity distribution thought the plant. The highest level of NR activity in P. vulgaris was clearly shown to occur in leaves in contrast with the lowest one detected in roots and nodules as widely seen for other tropical species of the Phaseoleae tribe. Conversely, P. lunatus had higher NR activity in the nodules, whereas its leaves exhibited a steadily decrease during the plant development. Indeed, at 32 days after emergence (pre-flowering stage), the nodulated P. vulgaris had approximately 95% of the total NR activity localized in its leaves, whereas in P. lunatus it was equally distributed in the nodules and in the leaves. Under long-term exposure to increasing exogenous level of nitrate, the leaf-NR activity of nodulated P. vulgaris presented a positive response, whereas the enzyme activity was very low and unresponsive in P. lunatus. In contrast, the nodule-NR activity showed a reverse response to the increasing NO(3)(-) level. The nodule-NR activity of P. lunatus significantly increased whereas in the P. vulgaris nodules it was very low and unresponsive. This present study suggests that P. lunatus inoculated with Rhizobium tropici presents a singular pattern of nitrate reduction distribution among leaves and nodules during the vegetative development. It is speculated that the nodulated Phaseolus lunatus may have different NR isoforms in their leaves (at least a constitutive type) and an inducible form in their nodules, responsive to long-term exposure to nitrate.     

Modification of paraheliotropic leaf movement in Phaseolus vulgaris by photon flux density

Abstract. The effects of modification of photon flux density (PFD, 400-700 nm) on paraheliotropic leaf movement were examined in Phaseolus vulgaris L. under controlled environmental conditions. The cosine of the angle of incidence to directional PFD (cos(i)), a measure of leaf movement, was linearly and negatively related to PFD. That is, leaflets progressively oriented away from a direct light beam in response to increasing PFD. The minimum PFD causing paraheliotropic movement was approximately 25 μmol m⁻² s⁻¹. When PFD was varied, tissue temperature changed due to an altered energy balance. Since a change in pulvinus temperature can affect leaf movement, experiments were conducted to distinguish the effects of PFD signal and pulvinis temperature. Leaflets oriented to reduce incident PFD levels in response to increasing PFD (either white light or blue light) when pulvinis temperature was kept constant. From these results, we conclude that changes in PFD signals alone can control paraheliotropic leaf movements. Phaseolus vulgaris grown outdoors oriented their leaflets to face towards the sun in the morning and again in late afternoon, but avoided the sun's direct rays at midday. This diurnal pattern of paraheliotropic leaf movements can be explained on the basis of known paraheliotropic movements in response to PFD and air temperature.     

Cold tolerance during larval development: effects on the thermal distribution limits of Leptinotarsa decemlineata

Insects' cold tolerance during their development is a surprisingly understudied subject in ecology, despite the fact that subzero temperatures during the growing season are common at high altitudes and latitudes. Subzero temperatures can have detrimental effects on organisms, restricting a species' range. This study addresses the question whether night frosts during the growing season have an instant or delayed negative impact on larval mortality of the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae). We also tested whether populations from the centre (Poland) and margins (Russia) of the distribution range of L. decemlineata differ in their responses to subzero exposure and a low rearing temperature. Larvae of three ages were subjected to a subzero temperature (−4 °C for 3 h simulating night frost) twice, after which they were reared on a fluctuating temperature regime of 10–15 °C. These rearing conditions imitated cool summer temperatures beyond the beetles' current range, such as in Finland. Individuals of both populations were highly cold tolerant, as only 3.1% of larvae died immediately following the subzero treatment. Nonetheless, the low rearing temperature was harmful to beetles of both populations. It caused high larval (ca. 90%) and overwintering (ca. 80%) mortality. As beetle performance was affected solely by rearing temperature, low temperatures during the growing season rather than night frosts apparently retard the beetle's northern expansion.     

Lectin and lectin-related proteins in Lima bean (Phaseolus lunatus L.) seeds: biochemical and evolutionary studies

Lectin-related polypeptides are a class of defence proteins found in seeds of Phaseolus species. In Lima bean (P. lunatus), these proteins and their genes have been well characterized in the Andean morphotype, which represents one of the two gene pools of this species. To study the molecular evolution of the lectin family in Lima bean we characterized the polypeptides belonging to this multigene family and cloned the genes belonging to the Mesoamerican gene pool. The latter gene pool contains components similar to those of the Andean pool, namely: an amylase inhibitor-like (AIL), an arcelin-like (ARL) lectin and the less abundant Lima bean lectin (LBL). These proteins originate from an ancestor gene of the lectin type which duplicated to yield the lectin gene and the progenitor of ARL and AIL. In this species. ARL represents an evolutionary intermediate form that precedes AIL. Phylogenetic analysis supports an Andean origin for Lima bean. The molecular evolutionary studies were extended to the genes of common bean and demonstrated that true lectin genes and the ancestor of lectin-related genes are the result of a duplication event that occurred before speciation. Lima and common bean followed different evolutionary pathways and in the latter species a second duplication event occurred that gave rise, in Mesoamerican wild genotypes, to arcelin genes.     

Free space iron pools in roots: generation and mobilization

A rapid and simple method for the determination of a ferric iron pool in the free space of roots is described. Formation of this pool depended on the source of iron in the nutrient solution. During growth in water culture at pH 5 to 6 with Fe-ethylenediaminetetraacetate, a free space pool of 500 to 1000 nanomoles Fe per gram fresh weight was formed in the roots of bean (Phaseolus vulgaris L. var. Prélude), maize (Zea mays L. var. Capella), and chlorophytum (Chlorophytum comosum [Thunb.] Jacques). No significant pool (less than 100 nanomoles per gram fresh weight) was formed with ferrioxamine. Upon impending Fe deficiency, bean and chlorophytum were able to mobilize this pool. Fe-deficient bean plants mobilized iron from the free space iron pool of another plant in the same vessel.     

VIRUSES OF COWPEA, VIGNA UNGUICULATA L. (WALP.), IN NIGERIA

The cowpea strain of tobacco mosaic virus was isolated from a range of leguminous hosts at Ibadan, but was rare in cultivated crops. Systemic symptoms in species infected experimentally are described.
A new virus of cowpea was also found in Nigeria. The physical properties (thermal inactivation point 56° C., dilution end-point 1/50,000 and longevity in vitro 4 days at 25° C.) differ from those for cowpea viruses reported elsewhere and the name cowpea yellow mosaic virus is proposed. This virus produces local lesions in French bean ( Phaseolus vulgaris L.) and local and systemic lesions in Bengal bean ( Mucuna aterrima Holland), but does not infect other leguminous hosts. The virus was purified and an antiserum prepared against it.
Both viruses are transmitted by a beetle ( Ootheca mutabilis Sahlb.) which loses infectivity within 48 hr. of leaving plants infected with either or both viruses.     

The influence of aluminium availability on phosphate uptake in Phaseolus vulgaris L. and Phaseolus lunatus L.

Aluminium toxicity is one of the major limiting factors of crop productivity on acid soils. High levels of available aluminium in soil may induce phosphorus deficiency in plants. This study investigates the influence of Aluminium (Al) on the phosphate (P_i) uptake of two Phaseolus species, Phaseolus vulgaris L. var. Red Kidney and Phaseolus lunatus L. The two bean species were treated first with solutions of Al at different concentrations (0, 25, 50 and 100 μM, pH 4.50) and second with solutions of P_i (150 μM) at pH 4.50. The higher the Al concentration the higher the Al concentration sorbed but P. vulgaris L var. Red Kidney adsorbed significantly more Al than P. lunatus L. Both species released organic acids: P. vulgaris L var. Red Kidney released fumaric acid and P. lunatus L. fumaric and oxalic acids which could have hindered further Al uptake.The two bean species showed a sigmoid P_i uptake trend but with two different mechanisms. P. vulgaris L var. Red Kidney showed a starting point of 3 h whereas P. lunatus L. adsorbed P_i immediately within the first minutes. In addition, P. vulgaris L var. Red Kidney presented significantly higher P_i uptake (higher uptake rate ‘k’ and higher maximum adsorption ‘a’ of the kinetic uptake model). The Al treatments did not significantly influence P_i uptake. Results suggest that P. lunatus L. might adopt an external Al detoxification mechanism by the release of oxalic acid. P. vulgaris L var. Red Kidney on the other hand seemed to adopt an internal detoxification mechanism even if the Al sorbed is poorly translocated into the shoots. More detailed studies will be necessary to better define Al tolerance and/or resistance of Phaseolus spp.